Syntheses and characterizations of the in vivo replicative bypass and mutagenic properties of the minor-groove O2-alkylthymidine lesions

Abstract

Endogenous metabolism, environmental exposure, and treatment with some chemotherapeutic agents can all give rise to DNA alkylation, which can occur on the phosphate backbone as well as the ring nitrogen or exocyclic nitrogen and oxygen atoms of nucleobases. Previous studies showed that the minor-groove O2-alkylated thymidine (O2-alkyldT) lesions are poorly repaired and persist in mammalian tissues. In the present study, we synthesized oligodeoxyribonucleotides harboring seven O2-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu or sBu, at a defined site and examined the impact of these lesions on DNA replication in Escherichia coli cells. Our results demonstrated that the replication bypass efficiencies of the O2-alkyldT lesions decreased with the chain length of the alkyl group, and these lesions directed promiscuous nucleotide misincorporation in E. coli cells. We also found that deficiency in Pol V, but not Pol II or Pol IV, led to a marked drop in bypass efficiencies for most O2-alkyldT lesions. We further showed that both Pol IV and Pol V were essential for the misincorporation of dCMP opposite these minor-groove DNA lesions, whereas only Pol V was indispensable for the T→A transversion introduced by these lesions. Depletion of Pol II, however, did not lead to any detectable alterations in mutation frequencies for any of the O2-alkyldT lesions. Thus, our study provided important new knowledge about the cytotoxic and mutagenic properties of the O2-alkyldT lesions and revealed the roles of the SOS-induced DNA polymerases in bypassing these lesions in E. coli cells.

Replicative Bypass of O2-Alkylthymidine Lesions in Vitro

“…Pol IV. 28, 29, 36, 39–41 This may be attributed to the fact that, unlike the N 2 -modified dG derivatives which still pair favorably with dCTP, the O 2 -alkyldT lesions do not have strong tendency to form Watson-Crick hydrogen bonding with any of the four canonical nucleotides. 28, 29, 36, 39–41 …”

“…28, 29 Human Pol η is known to accurately bypass thymine-thymine cyclobutane pyrimidine dimers (CPD) with a similar accuracy and efficiency as bypassing the corresponding unmodified nucleosides. 16–21 Pol η is unique among the Y-family polymerases due to its spacious active site which has the ability to accommodate the two cross-linked thymine bases in the CPD lesion.…”

Mass Spectrometry-Based Quantitative Strategies for Assessing the Biological Consequences and Repair of DNA Adducts

“…In addition, the incorporation of LC-MS/MS into the workflow enables accurate identification of mutant products arising from replicative or transcriptional bypass of DNA lesions. In this respect, the LC-MS/MS approach can be used to interrogate nucleotide misincorporation or indel events at or near the original lesion site rather than examining only the mutation at the lesion site in the restriction endonuclease and postlabeling (REAP) assay 3,6,9,11,12,15,20 . The MS-based methods are also quite sensitive and can be reliably used for determining a mutation frequency that is ~0.5%, with the standard deviation being ~0.1% that is comparable to the reproducibility of other methods such as the REAP assay 6,9,12,38 .…”

“…9,12-14,19,20,24 The experimental system begins with the ligation of lesion-bearing or lesion-free control ODNs into the EcoRI-linearized M13 genome with the help of two scaffolds flanking the lesion site (Figure 2a). The scaffolds and unligated linear plasmid DNA are degraded by the 3′✧5′ exonuclease activity of T4 DNA polymerase.…”

“…9,12 More recently, polyacrylamide gel electrophoresis (PAGE) has also been incorporated into the workflow of MS-based in vivo replication assay. 12-14,19,20,24 As an example, it was shown that the restriction fragments with a single nucleotide difference arising from replication of O 2 -EtdT-containing plasmid in E. coli cells can be readily resolved from each other with 30% native PAGE analysis. 19 In this context, PAGE analysis can be directly used for quantifying the restriction fragments of interest, while LC-MS and MS/MS are employed for the identifications of the restriction fragments.…”